Golden Gate Assembly system dedicated to complex pathway manipulation in Yarrowia lipolytica

Abstract

SummaryIn this study, we have adopted Golden Gate modular cloning strategy to develop a robust and versatile DNA assembly platform for the nonconventional yeast Yarrowia lipolytica. To this end, a broad set of destination vectors and interchangeable building blocks have been constructed. The DNA modules were assembled on a scaffold of predesigned 4 nt overhangs covering three transcription units (each bearing promoter, gene and terminator), selection marker gene and genomic integration targeting sequences, constituting altogether thirteen elements. Previously validated DNA modules (regulatory elements and selection markers) were adopted as the Golden Gate bricks. The system's operability was demonstrated based on synthetic pathway of carotenoid production. This technology greatly enriches a molecular biology toolbox dedicated to this industrially relevant microorganism enabling fast combinatorial cloning of complex synthetic pathways.

A synthetic biology approach to transform Yarrowia lipolytica into a competitive biotechnological producer of β‐carotene

“…Previous reports have proven that the overexpression of three genes, geranylgeranyl diphosphate synthase ( GGS1 from Y. lipolytica ), pytoene synthase/lycopene cyclase and phytoene dehydrogenase ( carPR and carB from Mucor circinelloides ) promote β‐carotene production in Y. lipolytica (Celinska et al, ; Gao et al, ). We here used an expression cassette (car‐cassette) where the expression of GGS1 is controlled by the promoter PGMp, of CarB by GAPDHp and of CarRP by TEF1p.…”

“…The construction of the car‐cassette with different promoters via traditional cloning would have been a very long and inefficient process. Thus, we took advantage of the recently developed Golden Gate toolbox for Y. lipolytica (Celinska et al, ) to perform a promoter shuffling strategy. The strategy, summarized in Figure , consists in a digestion‐ligation reaction guided by BsaI defined sites where the three promoters can be introduced in each of the three promoter positions in the car‐cassette.…”

“…The construction of carTEF‐cassette was done using the recently developed Golden Gate (GG) toolbox for Y. lipolytica (Celinska et al, ) based on (Engler & Marillonnet, ). Briefly, primers carrying pre‐designed 4‐nt overhangs and externally located BsaI recognition sites were designed and used to amplify the building blocks which were then cloned in donor vectors (Zero Blunt® TOPO® PCR Cloning Kit, Thermo Fisher Scientific).…”

A modular Golden Gate toolkit for Yarrowia lipolytica synthetic biology

“…The capacity and efficacy of our system to express three TUs on a same construct has been validated previously (Celinska et al ., ; Larroude et al ., ). However, the expression levels of the three TUs have not been evaluated.…”

Extraction and purification of violacein from Yarrowia lipolytica cells using aqueous solutions of surfactants

“…Genes were amplified from an Escherichia coli operon kindly provided by Cyrille Pauthenier . Constructions of the violacein VioABE and VioCD cassettes were done following the Golden Gate Assembly (GGA) strategy dedicated for Y. lipolytica according to Celińska et al . and Larroude et al .…”